The growth and propagation of mammalian cells in vitro (generically designated as tissue culture) is widely used in experimental biology and for the production of viral vaccines. In the future, tissue culture may find increasing use in the production of useful biological molecules generated by recombinant DNA techniques.
Typically, mammalian cells are cultured under conditions in which the hydrogen ion concentration (pH, the negative logarithm of the hydrogen ion concentration), temperature, humidity, osmolarity and concentration of certain ions are controlled within relatively narrow limits. Sterile conditions are necessary to exclude contamination and subsequent overgrowth by microorganisms.
In the vast majority of mammalian tissue culture systems in use at present, pH is maintained near physiologic levels (about pH 7.4) by utilizing a bicarbonate buffering system (H.multidot.HCO.sub.3 .revreaction.H.sup.+ +HCO.sub.3.sup.-) in the tissue culture fluid, in conjunction with an incubator in which carbon dioxide (CO.sub.2) is infused at a rate sufficient to maintain a concentration in the incubator atmosphere of approximately 5 to 7 volume percent. The CO.sub.2 reacts with water to form the weak acid, carbonic acid, which in turn interacts with bicarbonate ion (HCO.sub.3.sup.-) in the tissue culture fluid to form a buffering system which maintains the pH near physiologic levels. Entry of CO.sub.2 from the incubator into the tissue culture flask is generally achieved by utilizing a loosely fitting cap or stopper on the culture flask so that a small open space remains for exchange of gas between flask and incubator. The limitations to this current system of cell culture are apparent (1) susceptibility to contamination by microorganisms because of gas flow through the small but unobstructed space between cap and the flask; (2) a slow and variable rate of achieving pH equilibration by diffusion of CO.sub.2 through the loose-fitting cap.
In U.S. Pat. No. 3,870,602, issued on Mar. 11, 1975, a culture flask is disclosed in which the walls of the flask are made from an impact resistant polystyrene plastic which is permeable to water vapor, oxygen and carbon dioxide and impermeable to any microorganism or spores growing within the flask. This type of flask in which the walls are gas permeable is effective in preventing microorganisms from entering or exiting the flask while at the same time allowing for uniform and relatively fast equilibration between the atmosphere in the incubator and the flask.
Removal of the flask from the controlled atmosphere of the incubator is often required during growth and culturing of mammalian cells. The flasks are usually removed for inspection and/or treatment of the cells and culture fluids. It is important that the pH of the cell culture be maintained at the desired physiologic level while the flask is outside of the incubator. A problem arises when gas permeable flasks of the type described above are used in such tissue culture systems due to the rapid escape of carbon dioxide from the flask when it is removed from the controlled atmosphere of the incubator. This rapid loss of carbon dioxide through the gas permeable walls of the flask results in the pH of the cell culture rising rapidly to levels which are not suitable for optimum cell growth.
It would be desirable to provide an improved gas permeable filter flask which not only includes the features of providing rapid and uniform equilibration between the flask atmosphere and the incubator atmosphere, but also includes features which allow the culture flask to be removed from the controlled atmosphere of the incubator for reasonably long times without subjecting the cell culture to undesirable changes in the pH of the system.